Most microscopes use conventional incandescent lamps for illuminating stained biological samples. Incandescent lamps produce white light that is a combination of all colors in the visible spectrum (from about 400 nm to about 700 nm). Unfortunately, incandescent lamps produce considerable heat, have low energy efficiency, and must often be replaced frequently.
Light emitting diodes (LEDs) are known light sources that typically produce a single color, covering only a narrow band in the visible spectrum. For example, LEDs producing narrow bands of light centered on 450 nm, 525 nm, and 625 nm will appear to the human eye as blue, green, and red, respectively. Color is visualized by humans using specialized cells in the eye. In particular, the human eye contains three kinds of color receptor cells. These include so-called S-cones, M-cones, and L-cones, which sense Short, Medium, and Long wavelengths, respectively, of visible light. All three types of cone cells are sensitive to a wideband of wavelengths, but have different peak sensitivities. For example, S-cones are most sensitive around 420 nm (blue) while M-cones are sensitive around 534 nm (green), and L-cones are sensitive around 564 nm (red).
The eye essentially integrates the spectral function, producing three signals. One signal is the light intensity. Another differentiates blue light from yellow. Finally, the third separates yellow into red or green light. This integration of the spectrum averages colors together. Light from a yellow LED (590 nm) will appear the same to the eye as light from green and red LEDs combined (530 nm and 650 nm), because in both cases red and green are balanced and outweigh blue.
Combining different intensities of red, green, and blue light can create the appearance of nearly any color. For example, a microscope illumination system that uses separate colored red, green, and blue LEDs can be tuned to any color of illumination, depending on the operator's preferences.
In typical microscopic imaging applications, white light such as the light emitted from conventional incandescent bulbs is needed to illuminate the biological sample. Pathologists and others trained in viewing biological samples for disease states are familiar with analyzing samples illuminated with a broadband, incandescent light source. Attempts have been made to use LEDs to imitate the white light emitted from conventional incandescent sources. For example, single LEDs have been produced that generate a mixture of colored light to approximate white light. For example, in the aforementioned design, blue light is emitted from a gallium nitride diode semiconductor (at around 460 nm). Secondary light, in the range of about 550 nm to around 650 nm is emitted by a phosphor coating located inside a polymer jacket. The combination of wavelengths produces “white” light having a relatively high color temperature. A problem with LEDs of the type described above is that they are not good at producing light at relatively long wavelengths (e.g., red light).
In yet another design, light from red, green and blue LEDs is combined to produce illumination which appears to be white, but does not contain the full visible spectrum. In particular, the intensity of the illumination in the yellow band (around 565 nm to 590 nm) is very low. This gap in the yellow portion of the spectrum causes some stained cell samples to appear to be a different color than if they were illuminated with an incandescent lamp. This is problematic because the stained biological sample will appear different to a pathologist or other trained individual under the LED-based white light as compared to conventional incandescent white light. Pathologists, however, are typically trained on microscopes that use broad-band incandescent light sources. Different visual appearances may lead to confusion and misinterpretation of slide results. There thus is a need for an LED-based illumination source which produces little heat, has an extended lifetime, and produces colorimetric results similar to those of incandescent lamps.